Anthopleura anjunae anti-tumor peptide (AAP-H) is a pentapeptide from the sea anemone Anthopleura anjunae with an amino acid sequence of Tyr-Val-Pro-Gly-Pro that is obtained by alkaline protease enzymatic hydrolysis extraction. In this study, we investigated the inhibitory effects of AAP-H on prostate cancer DU-145 cell proliferation using a methylthiazolyldiphenyl-tetrazolium bromide assay. Cell morphology was analyzed by hematoxylin-eosin staining, acridine orange/ethidium bromide fluorescence staining, Hoechst 33258 fluorescence staining, and scanning electron microscopy. The mitochondrial membrane potential was determined by flow cytometry following JC-1 staining. The cell apoptosis rate was measured by Annexin V-fluorescein isothiocyanate and propidium iodide staining followed by flow cytometric analysis, and the expression of apoptosis-associated proteins was assayed by Western blotting. The results demonstrated that AAP-H induced significant reductions in the number of viable cells and increased cell death in both a dose-dependent and time-dependent manner, with an IC50 of approximately 9.605 mM, 7.910 mM, and 2.298 mM at 24 h, 48 h, and 72 h, respectively. The morphologic characteristics of apoptotic cells were observed after treatment with AAP-H. The mitochondrial membrane potential was markedly decreased, and apoptosis increased after AAP-H treatment. Pro-apoptotic proteins, such as Bax, cytochrome-C, caspase-3, and caspase-9 were increased, but Bcl-2 was decreased. These findings suggest that AAP-H has moderate inhibitory effects on prostate cancer DU-145 cells, and the mechanism might involve the mitochondria-mediated apoptotic pathway. Therefore, AAP-H is a candidate anti-prostate cancer drug or health-care food.
Keywords: Anthopleura anjunae; apoptosis rate; apoptosis-associated proteins; mitochondria membrane potential; morphology.